1. Field of the Invention
The present invention is directed to a method for the fluorescence spectroscopic determination of a biological marker containing bound lanthanide ions. More specifically, the invention is directed to such a method wherein the fluorescence of the lanthanide is enhanced by contacting the marker with a solution comprising a polyanion and an enhancer reagent, such that the lanthanide metal ions dissociate from the marker and reassociate with the enhancer reagent, thereby transforming the lanthanide metal ion into a highly fluorescent form. The amount of lanthanide metal ion which has been liberated from the marker and associated with the enhancer reagent then can be determined.
2. Prior Art
Lanthanide chelates are a preferred means of labeling detectors of biomolecules for time resolved fluorescence (TRF). A lanthanide metal ion, such as a europium or terbium ion, is coupled to a biological molecule of interest through a chelate-forming compound, such as ethylenediaminetetraacetic acid (EDTA) or an analogue thereof, and excited by a short radiation pulse. Detection of fluorescence of the marker is determined after the fluorescence from any background noise source has substantially ceased.
The lanthanide carrier-chelate can be either fluorescent or non-fluorescent. In either case, the lanthanide is rendered highly fluorescent via dissociation from the carrier chelate and subsequent enhancement through association with organic enhancer molecules.
One process that has been given commercial application is that disclosed in U.S. Pat. No. 4,565,790, issued to Hemmila et al. This patent teaches using an enhancer to dissociate the lanthanide from the carrier chelate by lowering the pH to less than 3.5. At such a low pH, the carrier chelate becomes protonated and no longer is capable of tightly binding the lanthanide. The lanthanide then can associate with a solution phase micellar organic enhancer and display intense fluorescence upon excitation.
Various literature references describe dissociative enhancers that work at acidic pH values or employ fluorescence-quenching amounts of urea and sodium dodecylsulfate (SDS). At pH values greater than 4, however, the lanthanide remains tightly bound and full fluorescent signal development may take hours or days.
An alternative process is that patented in U.S. Pat. No. 5,124,268, issued to Dakubu. In this method, an acid dissolution solution having a pH of about 2.0 is used to liberate the lanthanide. Then the addition of a higher pH solution containing enhancement reagents, such as .beta.-naphthoyltrifluoroacetone (.beta.-NTA), thenoyltrifluoroacetone (TTA), trioctylphosphineoxide (TOPO), etc., serves to neutralize the solution and allow association to the lanthanide with the enhancers in a fluorescent form.
Other methods for enhancement detection of lanthanide chelates use carrier chelates that directly excite the bound lanthanide (direct fluors). Exemplary compounds are found in U.S. Pat. Nos. 5,622,821 and 5,373,093. Such compounds can be used when localized signal information for the sample is important and/or when a homogeneous assay is required.
Although all of these methods can be useful, each has its drawbacks. The harsh conditions used for acid dissolution in the '790 patent usually disrupt association between the detector, capture reagent and analyte. The owners of the '790 patent, in literature regarding their commercial process, also have warned against using their enhancement solution with stronger chelates, such as diethylenetriamine pentacetic acid (DTPA). The procedure taught in the '268 patent does not suffer from these disadvantages, but is cumbersome in that it requires two sequential additions for detection and the signal generation is highly dependent on adding the correct amount of buffered enhancers to the acid portion. Undershooting or overshooting the desired final pH causes a reduction in the maximum fluorescence. In direct fluor methods, the fluorescence signal observed is about 20-1000 times less intense than the signal generated by enhancement reagents, limiting their use to assays where ultimate sensitivity is not required.
In view of the shortcomings of the known procedures, further improvements are sought.